Myostatin (also known as GDF-8) was discovered in 1997 as a novel cytokine belonging to the TGF-β superfamily. Its expression is specific to skeletal muscle which is a primary tissue responsible for movement and metabolism. Myostatin-deficient mutant animals show significant muscle hypertrophy where skeletal muscle mass is increased twice as much as in wild-type animals, so that myostatin is considered to be a negative control factor for skeletal muscle mass.
Based on the above findings, a therapeutic strategy can be designed to treat amyotrophic diseases or muscle wasting diseases through inhibition of myostatin. Skeletal muscle atrophy will induce not only limitation of daily living activities due to muscle weakness, but also serious systemic complications such as undernutrition and respiratory failure. Target diseases of this therapeutic strategy may include myogenic amyotrophy (e.g., muscular dystrophy, congenital myopathy, inclusion body myositis), neurogenic amyotrophy (e.g., amyotrophic lateral sclerosis, spinal muscular atrophy, spinal and bulbar muscular atrophy), disuse amyotrophy (e.g., apoplexy-induced disuse syndrome), muscle wasting diseases (e.g., cancer cachexia, sepsis-related amyotrophy), various types of sarcopenia including age-related skeletal muscle loss (age-related sarcopenia), etc.
The human myostatin gene is located on the long arm of chromosome 2. From three exons constituting this gene, mature mRNA having a chain length of approximately 2.8 kilobases is transcribed and further translated into a precursor polypeptide consisting of 375 amino acid residues. Myostatin precursor polypeptide molecules form a dimer through disulfide bonding between their C-terminal domains, and then cleaved between amino acid residues at positions 266 and 267 (R-D) in endoplasmic reticulum by the actin of a protease of the Furin family, so that the precursor dimer is divided into an N-terminal propeptide and a C-terminal domain dimer which will function later as active myostatin. These peptides are associated through non-covalent bonding and secreted as an inactive complex into the extracellular environment. This complex is further dissociated when the N-terminal propeptide is cleaved off between amino acid residues at positions 98 and 99 (R-D) by the action of a matrix metalloprotease of the BMP1/Tolloid family, whereby an active myostatin dimer appears.
In recent years, attention has been focused on antisense nucleic acid drugs, which are designed and chemically synthesized as short antisense artificial nucleic acids binding complementarily to a part of precursor mRNA in an attempt to inhibit mRNA function. In the normal mechanism of gene transcription, introns in precursor mRNA are cleaved and removed by the action of an enzyme complex called spliceosome to thereby generate mature mRNA. An antisense nucleic acid for exon skipping is designed to modify this spliceosome-mediated splicing regulatory mechanism and induce the generation of mRNA different from normal mature mRNA, thereby inhibiting the function of the gene. Moreover, mRNA is associated not only with the spliceosome, but also with an mRNA-stabilizing protein or an expression/translation regulatory factor (including miRNA) for regulation of mRNA degradation, expression and translation. An antisense nucleic acid is also considered to inhibit the association of such an mRNA-binding protein to its target mRNA, thereby inhibiting the function of the gene.
Currently, some antisense nucleic acids have been known to cause exon skipping in myostatin (Patent Documents 1 to 3 and Non-patent Documents 1 to 4).